We wish to understand how the nicotinic acetylcholine receptor functions as a ligand-gated ion channel and also to define the interactions within the post-synaptic memberane that are necessary to maintain acetylcholine receptors in high density clusters restricted to regions of the muscle plasma membrane directly underlying the nerve terminal. Torpedo electric tissue will be fractionated to isolate post-synaptic membranes to be used in studies of receptor structure and function. Functional domains within the receptor to be identified include the site of binding of agonists and competitive antagonists as well as the separate binding site for amine non-competitive antagonists. Binding sites will be labeled covalently by appropriate radiolabeled affinity reagents, and the receptor will be degraded to identify the labeled peptides in terms of the known amino acid sequences of the receptor subunits. To determine how the ligand binding sites are oriented within the three-dimensional structure of the receptor, biochemical and immunological labeling procedures will be used to identify receptor domains exposed at the extracellular surface, buried within the lipid bilayer, or exposed on the cytoplasmic surface. These techniques will also be used to identify regions of each receptor subunit in contact with each other or with non-receptor proteins of the post-synamptic membrane. Receptors in the isolated Torpedo post-synaptic membrane have very restricted motility unless the non-receptor peripheral proteins are removed. We will characterize by biochemical and immunological means the structure of the principal non-receptor protein (43kDa protein) and determine whether interactions between this protein and the receptor restrict motility of the receptor.